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1.
Dr. Reiko Ueoka Dr. Roy A. Meoded Alejandro Gran‐Scheuch Dr. Agneya Bhushan Prof. Dr. Marco W. Fraaije Prof. Dr. Jörn Piel 《Angewandte Chemie (International ed. in English)》2020,59(20):7761-7765
Bacterial trans‐acyltransferase polyketide synthases (trans‐AT PKSs) are multimodular megaenzymes that biosynthesize many bioactive natural products. They contain a remarkable range of domains and module types that introduce different substituents into growing polyketide chains. As one such modification, we recently reported Baeyer–Villiger‐type oxygen insertion into nascent polyketide backbones, thereby generating malonyl thioester intermediates. In this work, genome mining focusing on architecturally diverse oxidation modules in trans‐AT PKSs led us to the culturable plant symbiont Gynuella sunshinyii, which harbors two distinct modules in one orphan PKS. The PKS product was revealed to be lobatamide A, a potent cytotoxin previously only known from a marine tunicate. Biochemical studies show that one module generates glycolyl thioester intermediates, while the other is proposed to be involved in oxime formation. The data suggest varied roles of oxygenation modules in the biosynthesis of polyketide scaffolds and support the importance of trans‐AT PKSs in the specialized metabolism of symbiotic bacteria. 相似文献
2.
Toblerols: Cyclopropanol‐Containing Polyketide Modulators of Antibiosis in Methylobacteria
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Dr. Reiko Ueoka Dr. Miriam Bortfeld‐Miller Dr. Brandon I. Morinaka Prof. Dr. Julia A. Vorholt Prof. Dr. Jörn Piel 《Angewandte Chemie (International ed. in English)》2018,57(4):977-981
Trans‐AT polyketide synthases (PKSs) are a family of biosynthetically versatile modular type I PKSs that generate bioactive polyketides of impressive structural diversity. In this study, we detected, in the genome of several bacteria a cryptic, architecturally unusual trans‐AT PKS gene cluster which eluded automated PKS prediction. Genomic mining of one of these strains, the model methylotroph Methylobacterium extorquens AM1, revealed unique epoxide‐ and cyclopropanol‐containing polyketides named toblerols. Relative and absolute stereochemistry were determined by NMR experiments, chemical derivatization, and the comparison of CD data between the derivatized natural product and a synthesized model compound. Biosynthetic data suggest that the cyclopropanol moiety is generated by carbon–carbon shortening of a more extended precursor. Surprisingly, a knock‐out strain impaired in polyketide production showed strong inhibitory activity against other methylobacteria in contrast to the wild‐type producer. The activity was inhibited by complementation with toblerols, thus suggesting that these compounds modulate an as‐yet unknown methylobacterial antibiotic. 相似文献
3.
Acyl‐Chain Elongation Drives Ketosynthase Substrate Selectivity in trans‐Acyltransferase Polyketide Synthases
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Matthew Jenner Dr. José Pedro Afonso Hannah R. Bailey Sarah Frank Dr. Annette Kampa Prof. Dr. Jörn Piel Dr. Neil J. Oldham 《Angewandte Chemie (International ed. in English)》2015,54(6):1817-1821
Type I modular polyketide synthases (PKSs), which are responsible for the biosynthesis of many biologically active agents, possess a ketosynthase (KS) domain within each module to catalyze chain elongation. Acylation of the KS active site Cys residue is followed by transfer to malonyl‐ACP to yield an extended β‐ketoacyl chain (ACP=acyl carrier protein). To date, the precise contribution of KS selectivity in controlling product fidelity has been unclear. Six KS domains from trans‐acyltransferase (trans‐AT) PKSs were subjected to a mass spectrometry based elongation assay, and higher substrate selectivity was identified for the elongating step than in preceding acylation. A close correspondence between the observed KS selectivity and that predicted by phylogenetic analysis was seen. These findings provide insights into the mechanism of KS selectivity in this important group of PKSs, can serve as guidance for engineering, and show that targeted mutagenesis can be used to expand the repertoire of acceptable substrates. 相似文献
4.
Eveline Vriens Dr. Dries De Ruysscher Dr. Angus N. M. Weir Dr. Sofie Dekimpe Dr. Gert Steurs Ahmed Shemy Leentje Persoons Dr. Ana Rita Santos Dr. Christopher Williams Prof. Dirk Daelemans Prof. Matthew P. Crump Prof. Arnout Voet Prof. Wim De Borggraeve Prof. Eveline Lescrinier Prof. Joleen Masschelein 《Angewandte Chemie (International ed. in English)》2023,62(34):e202304476
Bacterial trans-acyltransferase polyketide synthases (trans-AT PKSs) are modular megaenzymes that employ unusual catalytic domains to assemble diverse bioactive natural products. One such PKS is responsible for the biosynthesis of the oximidine anticancer agents, oxime-substituted benzolactone enamides that inhibit vacuolar H+-ATPases. Here, we describe the identification of the oximidine gene cluster in Pseudomonas baetica and the characterization of four novel oximidine variants, including a structurally simpler intermediate that retains potent anticancer activity. Using a combination of in vivo, in vitro and computational approaches, we experimentally elucidate the oximidine biosynthetic pathway and reveal an unprecedented mechanism for O-methyloxime formation. We show that this process involves a specialized monooxygenase and methyltransferase domain and provide insight into their activity, mechanism and specificity. Our findings expand the catalytic capabilities of trans-AT PKSs and identify potential strategies for the production of novel oximidine analogues. 相似文献
5.
Dr. Reiko Ueoka Dr. Roy A. Meoded Alejandro Gran-Scheuch Dr. Agneya Bhushan Prof. Dr. Marco W. Fraaije Prof. Dr. Jörn Piel 《Angewandte Chemie (Weinheim an der Bergstrasse, Germany)》2020,132(20):7835-7839
Bacterial trans-acyltransferase polyketide synthases (trans-AT PKSs) are multimodular megaenzymes that biosynthesize many bioactive natural products. They contain a remarkable range of domains and module types that introduce different substituents into growing polyketide chains. As one such modification, we recently reported Baeyer–Villiger-type oxygen insertion into nascent polyketide backbones, thereby generating malonyl thioester intermediates. In this work, genome mining focusing on architecturally diverse oxidation modules in trans-AT PKSs led us to the culturable plant symbiont Gynuella sunshinyii, which harbors two distinct modules in one orphan PKS. The PKS product was revealed to be lobatamide A, a potent cytotoxin previously only known from a marine tunicate. Biochemical studies show that one module generates glycolyl thioester intermediates, while the other is proposed to be involved in oxime formation. The data suggest varied roles of oxygenation modules in the biosynthesis of polyketide scaffolds and support the importance of trans-AT PKSs in the specialized metabolism of symbiotic bacteria. 相似文献
6.
Dr. Hannah A. Minas Romain M. M. François Dr. Franziska Hemmerling Dr. Amy E. Fraley Cora L. Dieterich Dr. Simon H. Rüdisser Dr. Roy A. Meoded Sabrina Collin Prof. Dr. Kira J. Weissman Dr. Arnaud Gruez Prof. Dr. Jörn Piel 《Angewandte Chemie (International ed. in English)》2023,62(34):e202304481
Modular trans-acyltransferase polyketide synthases (trans-AT PKSs) are enzymatic assembly lines that biosynthesize complex polyketide natural products. Relative to their better studied cis-AT counterparts, the trans-AT PKSs introduce remarkable chemical diversity into their polyketide products. A notable example is the lobatamide A PKS, which incorporates a methylated oxime. Here we demonstrate biochemically that this functionality is installed on-line by an unusual oxygenase-containing bimodule. Furthermore, analysis of the oxygenase crystal structure coupled with site-directed mutagenesis allows us to propose a model for catalysis, as well as identifying key protein-protein interactions that support this chemistry. Overall, our work adds oxime-forming machinery to the biomolecular toolbox available for trans-AT PKS engineering, opening the way to introducing such masked aldehyde functionalities into diverse polyketides. 相似文献
7.
Jensen K Niederkrüger H Zimmermann K Vagstad AL Moldenhauer J Brendel N Frank S Pöplau P Kohlhaas C Townsend CA Oldiges M Hertweck C Piel J 《Chemistry & biology》2012,19(3):329-339
Trans-acyltransferase polyketide synthases (trans-AT PKSs) are an important group of bacterial enzymes producing bioactive polyketides. One difference from textbook PKSs is the presence of one or more free-standing AT-like enzymes. While one homolog loads the PKS with malonyl units, the function of the second copy (AT2) was unknown. We studied the two ATs PedC and PedD involved in pederin biosynthesis in an uncultivated symbiont. PedD displayed malonyl- but not acetyltransferase activity toward various acyl carrier proteins (ACPs). In contrast, the AT2 PedC efficiently hydrolyzed acyl units bound to N-acetylcysteamine or ACP. It accepted substrates with various chain lengths and functionalizations but did not cleave malonyl-ACP. These data are consistent with the role of PedC in?PKS proofreading, suggesting a similar function for other AT2 homologs and providing strategies for?polyketide titer improvement and biosynthetic investigations. 相似文献
8.
Iterative Assembly of Two Separate Polyketide Chains by the Same Single‐Module Bacterial Polyketide Synthase in the Biosynthesis of HSAF
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Dr. Yaoyao Li Haotong Chen Yanjiao Ding Yunxuan Xie Dr. Haoxin Wang Prof. Dr. Ronald L. Cerny Prof. Dr. Yuemao Shen Prof. Dr. Liangcheng Du 《Angewandte Chemie (International ed. in English)》2014,53(29):7524-7530
Antifungal HSAF (heat‐stable antifungal factor, dihydromaltophilin) is a polycyclic tetramate macrolactam from the biocontrol agent Lysobacter enzymogenes. Its biosynthetic gene cluster contains only a single‐module polyketide synthase–nonribosomal peptide synthetase (PKS‐NRPS), although two separate hexaketide chains are required to assemble the skeleton. To address the unusual biosynthetic mechanism, we expressed the biosynthetic genes in two “clean” strains of Streptomyces and showed the production of HSAF analogues and a polyene tetramate intermediate. We then expressed the PKS module in Escherichia coli and purified the enzyme. Upon incubation of the enzyme with acyl‐coenzyme A and reduced nicotinamide adenine dinucleotide phosphate (NADPH), a polyene was detected in the tryptic acyl carrier protein (ACP). Finally, we incubated the polyene–PKS with the NRPS module in the presence of ornithine and adenosine triphosphate (ATP), and we detected the same polyene tetramate as that in Streptomyces transformed with the PKS‐NRPS alone. Together, our results provide evidence for an unusual iterative biosynthetic mechanism for bacterial polyketide–peptide natural products. 相似文献
9.
Danyao Du Dr. Yohei Katsuyama Dr. Kazuo Shin‐ya Dr. Yasuo Ohnishi 《Angewandte Chemie (International ed. in English)》2018,57(7):1954-1957
While type II polyketide synthases (PKSs) are known for producing aromatic compounds, a phylogenetically new subfamily of type II PKSs have been recently proposed to synthesize polyene structures. Here we report in vitro analysis of such a type II PKS, IgaPKS for ishigamide biosynthesis. The ketoreductase (Iga13) and dehydratase (Iga16) were shown to catalyze the reduction of a β‐keto group and dehydration of a β‐hydroxy group, respectively, to form a trans double bond. Incubation of the acyl carrier protein (Iga10), the ketosynthase/chain length factor complex (Iga11–Iga12), Iga13 and Iga16 with malonyl and hexanoyl‐CoAs and NADPH followed by KOH hydrolysis resulted in the formation of four unsaturated carboxylic acids (C8, C10, C12, and C14), indicating that IgaPKS catalyzes tetraene formation by repeating the cycle of condensation, keto‐reduction and dehydration with strict stereo‐specificity. We propose “highly reducing type II PKS subfamily” for the polyene‐producing type II PKSs. 相似文献
10.
Douglas R. Cohen Prof. Craig A. Townsend 《Angewandte Chemie (International ed. in English)》2018,57(20):5650-5654
Despite the identification of a β‐hydroxyhexaene produced by the enediyne polyketide synthases (PKSs), the post‐PKS biosynthetic steps to the individual members of this antitumor and antibiotic family remain largely unknown. The massive biosynthetic gene clusters (BGCs) that direct the formation of each product caution that many steps could be required. It was recently demonstrated that the enediyne PKS in the dynemicin A BGC from Micromonospora chersina gives rise to both the anthraquinone and enediyne halves of the molecule. We now present the first evidence for a mid‐pathway intermediate in dynemicin A biosynthesis, an iodoanthracene bearing a fused thiolactone, which was shown to be incorporated selectively into the final product. This unusual precursor reflects just how little is understood about these biosynthetic pathways, yet constrains the mechanisms that can act to achieve the key heterodimerization to the anthraquinone‐containing subclass of enediynes. 相似文献
11.
Structural Basis of a Broadly Selective Acyltransferase from the Polyketide Synthase of Splenocin
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Yuan Li Wan Zhang Hui Zhang Wenya Tian Lian Wu Shuwen Wang Mengmeng Zheng Jinru Zhang Chenghai Sun Prof. Zixin Deng Prof. Yuhui Sun Prof. Xudong Qu Prof. Jiahai Zhou 《Angewandte Chemie (International ed. in English)》2018,57(20):5823-5827
Polyketides are a large family of pharmaceutically important natural products, and the structural modification of their scaffolds is significant for drug development. Herein, we report high‐resolution X‐ray crystal structures of the broadly selective acyltransferase (AT) from the splenocin polyketide synthase (SpnD‐AT) in the apo form and in complex with benzylmalonyl and pentynylmalonyl extender unit mimics. These structures revealed the molecular basis for the stereoselectivity and substrate specificity of SpnD‐AT, and enabled the engineering of the industrially important Ery‐AT6 to broaden its substrate scope to include three new types of extender units. 相似文献
12.
Yi Tang 《Tetrahedron》2004,60(35):7659-7671
Polycyclic aromatic polyketides such as actinorhodin and tetracenomycin are synthesized from acetate equivalents by type II polyketide synthases (PKS). Their carbon chain backbones are derived from malonyl-CoA building blocks through the action of a minimal PKS module consisting of a ketosynthase, a chain length factor, an acyl carrier protein (ACP) and a malonyl-CoA/ACP transacylase. In contrast to these acetogenic polyketides, the backbones of a few aromatic polyketide natural products, such as the R1128 antibiotics, are primed by non-acetate building blocks. These polyketides are synthesized by bimodular PKSs comprising of a dedicated initiation module, which includes a ketosynthase, acyl transferase and ACP, as well as a minimal PKS module. Recently we showed that regioselectively modified polyketides could be synthesized through the genetic recombination of initiation modules and minimal PKS modules from different polyketide biosynthetic pathways (Tang et al. PLoS Biol. 2004, 2, 227-238). For example, the actinorhodin and tetracenomycin minimal PKSs could accept and elongate unnatural primer units from the R1128 initiation module. In this report we provide further examples of using heterologous bimodular PKSs for the engineered biosynthesis of new aromatic polyketides. In addition to providing insights into the biosynthetic mechanisms of aromatic PKSs, our findings also highlight considerable potential for crosstalk between amino acid catabolism and aromatic polyketide biosynthesis. For example, exogenously supplied unnatural amino acids are efficiently incorporated into bioactive anthraquinone antibiotics. 相似文献
13.
Dr. Elena Riva Dr. Ina Wilkening Silvia Gazzola W. M. Ariel Li Dr. Luke Smith Prof. Peter F. Leadlay Dr. Manuela Tosin 《Angewandte Chemie (International ed. in English)》2014,53(44):11944-11949
A library of functionalized chemical probes capable of reacting with ketosynthase‐bound biosynthetic intermediates was prepared and utilized to explore in vivo polyketide diversification. Fermentation of ACP mutants of S. lasaliensis in the presence of the probes generated a range of unnatural polyketide derivatives, including novel putative lasalocid A derivatives characterized by variable aryl ketone moieties and linear polyketide chains (bearing alkyne/azide handles and fluorine) flanking the polyether scaffold. By providing direct information on microorganism tolerance and enzyme processing of unnatural malonyl‐ACP analogues, as well as on the amenability of unnatural polyketides to further structural modifications, the chemical probes constitute invaluable tools for the development of novel mutasynthesis and synthetic biology. 相似文献
14.
Dr. Ashley J. Winter Dr. R. Nisha Khanizeman Dr. Abigail M. C. Barker-Mountford Dr. Andrew J. Devine Dr. Luoyi Wang Dr. Zhongshu Song Dr. Jonathan A. Davies Prof. Paul R. Race Dr. Christopher Williams Prof. Thomas J. Simpson Prof. Christine L. Willis Prof. Matthew P. Crump 《Angewandte Chemie (International ed. in English)》2023,62(47):e202312514
Mupirocin is a clinically important antibiotic produced by a trans-AT Type I polyketide synthase (PKS) in Pseudomonas fluorescens. The major bioactive metabolite, pseudomonic acid A (PA−A), is assembled on a tetrasubstituted tetrahydropyran (THP) core incorporating a 6-hydroxy group proposed to be introduced by α-hydroxylation of the thioester of the acyl carrier protein (ACP) bound polyketide chain. Herein, we describe an in vitro approach combining purified enzyme components, chemical synthesis, isotopic labelling, mass spectrometry and NMR in conjunction with in vivo studies leading to the first characterisation of the α-hydroxylation bimodule of the mupirocin biosynthetic pathway. These studies reveal the precise timing of hydroxylation by MupA, substrate specificity and the ACP dependency of the enzyme components that comprise this α-hydroxylation bimodule. Furthermore, using purified enzyme, it is shown that the MmpA KS0 shows relaxed substrate specificity, suggesting precise spatiotemporal control of in trans MupA recruitment in the context of the PKS. Finally, the detection of multiple intermodular MupA/ACP interactions suggests these bimodules may integrate MupA into their assembly. 相似文献
15.
Zirong Deng Jing Liu Tong Li Huayue Li Zengzhi Liu Yujing Dong Wenli Li 《Angewandte Chemie (International ed. in English)》2021,60(1):153-158
As a unique structural moiety in natural products, cinnamoyl lipids (CLs), are proposed to be assembled by unusual type II polyketide synthases (PKSs). Herein, we demonstrate that the assembly of the CL compounds youssoufenes is accomplished by a PKS system that uniquely harbors three phylogenetically different ketosynthase/chain length factor (KS/CLF) complexes (YsfB/C, YsfD/E, and YsfJ/K). Through in vivo gene inactivation and in vitro reconstitution, as well as an intracellular tagged carrier‐protein tracking (ITCT) strategy developed in this study, we successfully elucidated the isomerase‐dependent ACP‐tethered polyunsaturated chain elongation process. The three KS/CLFs were revealed to modularly assemble different parts of the youssoufene skeleton, during which benzene ring closure happens right after the formation of an ACP‐tethered C18 polyene. Of note, the ITCT strategy could significantly contribute to the elucidation of other carrier‐protein‐dependent biosynthetic machineries. 相似文献
16.
Paenilamicin: Structure and Biosynthesis of a Hybrid Nonribosomal Peptide/Polyketide Antibiotic from the Bee Pathogen Paenibacillus larvae
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Sebastian Müller Dr. Eva Garcia‐Gonzalez Dr. Andi Mainz Gillian Hertlein Nina C. Heid Eva Mösker Hans van den Elst Prof. Dr. Herman S. Overkleeft Priv.‐Doz. Dr. Elke Genersch Prof.Dr. Roderich D. Süssmuth 《Angewandte Chemie (International ed. in English)》2014,53(40):10821-10825
The spore‐forming bacterium Paenibacillus larvae is the causative agent of American Foulbrood (AFB), a fatal disease of honey bees that occurs worldwide. Previously, we identified a complex hybrid nonribosomal peptide/polyketide synthesis (NRPS/PKS) gene cluster in the genome of P. larvae. Herein, we present the isolation and structure elucidation of the antibacterial and antifungal products of this gene cluster, termed paenilamicins. The unique structures of the paenilamicins give deep insight into the underlying complex hybrid NRPS/PKS biosynthetic machinery. Bee larval co‐infection assays reveal that the paenilamicins are employed by P. larvae in fighting ecological niche competitors and are not directly involved in killing the bee larvae. Their antibacterial and antifungal activities qualify the paenilamicins as attractive candidates for drug development. 相似文献
17.
Samantha L. Kilgour Robert Jenkins Dr. Manuela Tosin 《Chemistry (Weinheim an der Bergstrasse, Germany)》2019,25(72):16511-16514
A photolabile carba(dethia) malonyl N-acetylcysteamine derivative was devised and prepared for the trapping of biosynthetic polyketide intermediates following light activation. From the lasalocid A polyketide assembly in a mutant strain of the soil bacterium Streptomyces lasaliensis, a previously undetected cyclised intermediate was identified and characterised, providing a new outlook on the timing of substrate processing. 相似文献
18.
Matthias Häckh Prof. Dr. Michael Müller Dr. Steffen Lüdeke 《Chemistry (Weinheim an der Bergstrasse, Germany)》2013,19(27):8922-8928
The stereospecificity of an enzymatic reaction depends on the way in which a substrate and its enantiomer bind to the active site. These binding modes cannot be easily predicted. We have studied the stereospecificity and stereoselectivity of the ketoreductase domain Tyl‐KR1 of the tylactone polyketide synthase from Streptomyces fradiae by analysing the stereochemical outcome of the reduction of five different keto ester substrates. The absolute configuration of the Tyl‐KR1 reduction products was determined by using vibrational circular dichroism (VCD) spectroscopy combined with quantum chemical calculations. The conversion of only one of the tested substrates, 2‐methyl‐3‐oxovaleric acid N‐acetylcysteamine thioester, afforded the expected anti‐(2R,3R) configuration of the α‐methyl‐β‐hydroxyl ester product, representing the stereochemistry observed for the physiological polyketide product tylactone. For all other substrates, which were modified with respect to the type of ester and/or the chain length (C4 instead of C5), the opposite configuration (anti‐(2S,3S)) was obtained with significant enantio‐ and diastereoselectivity. Inversion of both stereocentres suggests completely different binding modes invoked by only minor modifications of the substrate structure. 相似文献
19.
Genome Mining and Comparative Biosynthesis of Meroterpenoids from Two Phylogenetically Distinct Fungi
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Xuan Zhang Ting Ting Wang Qin Lan Xu Ying Xiong Li Zhang Hao Han Kuang Xu Dr. Wen Jie Guo Prof. Qiang Xu Prof. Ren Xiang Tan Prof. Hui Ming Ge 《Angewandte Chemie (International ed. in English)》2018,57(27):8184-8188
20.
Divergent Mechanistic Routes for the Formation of gem‐Dimethyl Groups in the Biosynthesis of Complex Polyketides
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Sean Poust Dr. Ryan M. Phelan Dr. Kai Deng Dr. Leonard Katz Dr. Christopher J. Petzold Prof. Dr. Jay D. Keasling 《Angewandte Chemie (International ed. in English)》2015,54(8):2370-2373
The gem‐dimethyl groups in polyketide‐derived natural products add steric bulk and, accordingly, lend increased stability to medicinal compounds, however, our ability to rationally incorporate this functional group in modified natural products is limited. In order to characterize the mechanism of gem‐dimethyl group formation, with a goal toward engineering of novel compounds containing this moiety, the gem‐dimethyl group producing polyketide synthase (PKS) modules of yersiniabactin and epothilone were characterized using mass spectrometry. The work demonstrated, contrary to the canonical understanding of reaction order in PKSs, that methylation can precede condensation in gem‐dimethyl group producing PKS modules. Experiments showed that both PKSs are able to use dimethylmalonyl acyl carrier protein (ACP) as an extender unit. Interestingly, for epothilone module 8, use of dimethylmalonyl‐ACP appeared to be the sole route to form a gem‐dimethylated product, while the yersiniabactin PKS could methylate before or after ketosynthase condensation. 相似文献